Dataset Information

AS precipitation and protein A affinity chromatography for abundant plasma protein depletion

ABSTRACT: Plasma is a highly valuable resource for biomarker research since it is easy obtainable and contains a high amount of information on patient health status. Although advancements in the field of proteomics enabled analysis of the plasma proteome, identification of low abundant proteins remains challenging due to high complexity and large dynamic range. In order to reduce the dynamic range of protein concentrations, a tandem depletion technique consisting of ammonium sulfate precipitation and Protein A affinity chromatography was developed. Using this method, 50 % of albumin, together with other high abundant proteins such as alpha-1-antitrypin, was depleted from the plasma sample at 20 % to 40 % ammonium sulfate saturation levels. In combination with immunoglobulin removal using a Protein A column, this technique delivered up to 40 new low- to medium abundance protein identifications when performing a shotgun mass spectrometry analysis. Compared to non-depleted plasma, 270 additional protein spots were observed during 2D-PAGE analysis. These results illustrate that this tandem depletion method is equivalent to commercial kits which are based on immune-affinity chromatography. Moreover, this method using protein A immunoglobulin depletion was shown to be highly reproducible and a minimal amount of non-target proteins was depleted. The combination of ammonium sulfate precipitation and Protein A affinity chromatography offers a low cost, efficient, straightforward and reproducible alternative to commercial kits, with proteins remaining in native conformation, allowing protein activity and protein interaction studies.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Blood Plasma

SUBMITTER: Lentel Pringels

LAB HEAD: Liliane Schoofs

PROVIDER: PXD008969 | Pride | 2018-05-08

REPOSITORIES: Pride

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Publications

Depletion of highly abundant proteins in blood plasma by ammonium sulfate precipitation for 2D-PAGE analysis.

Mahn Andrea A Ismail Maritza M

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 20110918 30

Ammonium sulfate precipitation (ASP) was explored as a method for depleting some highly abundant proteins from blood plasma, in order to reduce the dynamic range of protein concentration and to improve the detection of low abundance proteins by 2D-PAGE. 40% ammonium sulfate saturation was chosen since it allowed depleting 39% albumin and 82% α-1-antitrypsin. ASP-depletion showed high reproducibility in 2D-PAGE analysis (4.2% variation in relative abundance of albumin), similar to that offered by ...[more]

PMID: 21963274

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Project description:Background. Chronic renal failure is characterized by progressive renal scarring and accelerated cardiovascular disease. In animal models, this is thought to be due to non-dialyzable uremic toxins M-bM-^@M-^S small, protein-bound molecules normally secreted via Organic Anion Transporters (OATs) in the proximal renal tubule,rather than filtered at the glomerulus. The best studied of these is indoxyl sulfate (IS). Methods. We examined global gene expression responses in cultured normal human renal tubular cells incubated with control plasma (n=5) or pre- and post-dialysis uremic plasma (n=10). Results. GeneSpring analysis of microarrays revealed significantly altered expression of 2016 genes. The expression of 537 genes M-bM-^@M-^\normalizedM-bM-^@M-^] in post-dialysis plasma, suggesting removal of M-bM-^@M-^\low molecular weight uremic toxinsM-bM-^@M-^] by dialysis. The expression of the majority of dysregulated genes (1479) was not normalized in post-dialysis plasma. These likely represent the effects of substances not effectively removed by dialysis (M-bM-^@M-^\protein-bound uremic toxinsM-bM-^@M-^]). Addition of indoxyl sulfateto control plasma simulated most effects (81.1%) of uremic plasma, while the addition of probenecid, an OAT inhibitor, to uremic plasma reversed most changes in gene expression. Analysis of molecular programs with the Gene Set Enrichment Analysis and the DAVID database revealed patterns common to indoxyl sulfate-treated control plasma, pre-dialysis, and post-dialysis uremic plasma. These included increased cell cycle, pro-inflammatory, and pro-fibrotic molecular programs, particularly genes in the TGF-M-NM-2 pathway. Conclusion: These findings provide insight into the role of non-dialyzable, protein-bound uremic toxins in the pathogenesis of renal scarring and uremic vasculopathy. The GSEA patterns confirm that inflammatory and fibrotic programs are active. Transcriptomic remodeling in response to external stimulus RNA from non-immortalized cultured renal cortical cells incubated with plasma samples from 5 controls, 10 uremics pre-dialysis, 10 uremics post-dialysis, 5 controls with indoxyl sulfate added, 5 uremics pre-dialysis with probenecid added, 5 uremics post-dialysis with indoxyl sulfate added

Dataset Information

AS precipitation and protein A affinity chromatography for abundant plasma protein depletion

Publications

Depletion of highly abundant proteins in blood plasma by ammonium sulfate precipitation for 2D-PAGE analysis.

Similar Datasets

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